Brien P. Riley

Identification of risk loci with shared effects on five major psychiatric disorders: a genome-wide analysis

BACKGROUND: Findings from family and twin studies suggest that genetic contributions to psychiatric disorders do not in all cases map to present diagnostic categories. We aimed to identify specific variants underlying genetic effects shared between the five disorders in the Psychiatric Genomics Consortium: autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and schizophrenia. METHODS: We analysed genome-wide single-nucleotide polymorphism (SNP) data for the five disorders in 33,332 cases and 27,888 controls of European ancestory. To characterise allelic effects on each disorder, we applied a multinomial logistic regression procedure with model selection to identify the best-fitting model of relations between genotype and phenotype. We examined cross-disorder effects of genome-wide significant loci previously identified for bipolar disorder and schizophrenia, and used polygenic risk-score analysis to examine such effects from a broader set of common variants. We undertook pathway analyses to establish the biological associations underlying genetic overlap for the five disorders. We used enrichment analysis of expression quantitative trait loci (eQTL) data to assess whether SNPs with cross-disorder association were enriched for regulatory SNPs in post-mortem brain-tissue samples. FINDINGS: SNPs at four loci surpassed the cutoff for genome-wide significance (p<5x10(-8)) in the primary analysis: regions on chromosomes 3p21 and 10q24, and SNPs within two L-type voltage-gated calcium channel subunits, CACNA1C and CACNB2. Model selection analysis supported effects of these loci for several disorders. Loci previously associated with bipolar disorder or schizophrenia had variable diagnostic specificity. Polygenic risk scores showed cross-disorder associations, notably between adult-onset disorders. Pathway analysis supported a role for calcium channel signalling genes for all five disorders. Finally, SNPs with evidence of cross-disorder association were enriched for brain eQTL markers. INTERPRETATION: Our findings show that specific SNPs are associated with a range of psychiatric disorders of childhood onset or adult onset. In particular, variation in calcium-channel activity genes seems to have pleiotropic effects on psychopathology. These results provide evidence relevant to the goal of moving beyond descriptive syndromes in psychiatry, and towards a nosology informed by disease cause. FUNDING: National Institute of Mental Health.BACKGROUND Findings from family and twin studies suggest that genetic contributions to psychiatric disorders do not in all cases map to present diagnostic categories. We aimed to identify specific variants underlying genetic effects shared between the five disorders in the Psychiatric Genomics Consortium: autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and schizophrenia. METHODS We analysed genome-wide single-nucleotide polymorphism (SNP) data for the five disorders in 33,332 cases and 27,888 controls of European ancestory. To characterise allelic effects on each disorder, we applied a multinomial logistic regression procedure with model selection to identify the best-fitting model of relations between genotype and phenotype. We examined cross-disorder effects of genome-wide significant loci previously identified for bipolar disorder and schizophrenia, and used polygenic risk-score analysis to examine such effects from a broader set of common variants. We undertook pathway analyses to establish the biological associations underlying genetic overlap for the five disorders. We used enrichment analysis of expression quantitative trait loci (eQTL) data to assess whether SNPs with cross-disorder association were enriched for regulatory SNPs in post-mortem brain-tissue samples. FINDINGS SNPs at four loci surpassed the cutoff for genome-wide significance (p<5×10(-8)) in the primary analysis: regions on chromosomes 3p21 and 10q24, and SNPs within two L-type voltage-gated calcium channel subunits, CACNA1C and CACNB2. Model selection analysis supported effects of these loci for several disorders. Loci previously associated with bipolar disorder or schizophrenia had variable diagnostic specificity. Polygenic risk scores showed cross-disorder associations, notably between adult-onset disorders. Pathway analysis supported a role for calcium channel signalling genes for all five disorders. Finally, SNPs with evidence of cross-disorder association were enriched for brain eQTL markers. INTERPRETATION Our findings show that specific SNPs are associated with a range of psychiatric disorders of childhood onset or adult onset. In particular, variation in calcium-channel activity genes seems to have pleiotropic effects on psychopathology. These results provide evidence relevant to the goal of moving beyond descriptive syndromes in psychiatry, and towards a nosology informed by disease cause. FUNDING National Institute of Mental Health.

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia

Left–right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Addictions Biology: Haplotype-Based Analysis for 130 Candidate Genes on a Single Array

AIMS To develop a panel of markers able to extract full haplotype information for candidate genes in alcoholism, other addictions and disorders of mood and anxiety. METHODS A total of 130 genes were haplotype tagged and genotyped in 7 case/control populations and 51 reference populations using Illumina GoldenGate SNP genotyping technology, determining haplotype coverage. We also constructed and determined the efficacy of a panel of 186 ancestry informative markers. RESULTS An average of 1465 loci were genotyped at an average completion rate of 91.3%, with an average call rate of 98.3% and replication rate of 99.7%. Completion and call rates were lowered by the performance of two datasets, highlighting the importance of the DNA quality in high throughput assays. A comparison of haplotypes captured by the Addictions Array tagging SNPs and commercially available whole-genome arrays from Illumina and Affymetrix shows comparable performance of the tag SNPs to the best whole-genome array in all populations for which data are available. CONCLUSIONS Arrays of haplotype-tagged candidate genes, such as this addictions-focused array, represent a cost-effective approach to generate high-quality SNP genotyping data useful for the haplotype-based analysis of panels of genes such as these 130 genes of interest to alcohol and addictions researchers. The inclusion of the 186 ancestry informative markers allows for the detection and correction for admixture and further enhances the utility of the array.

Fine mapping of ZNF804A and genome-wide significant evidence for its involvement in schizophrenia and bipolar disorder

A recent genome-wide association study (GWAS) reported evidence for association between rs1344706 within ZNF804A (encoding zinc-finger protein 804A) and schizophrenia (P=1.61 × 10−7), and stronger evidence when the phenotype was broadened to include bipolar disorder (P=9.96 × 10−9). In this study we provide additional evidence for association through meta-analysis of a larger data set (schizophrenia/schizoaffective disorder N=18 945, schizophrenia plus bipolar disorder N=21 274 and controls N=38 675). We also sought to better localize the association signal using a combination of de novo polymorphism discovery in exons, pooled de novo polymorphism discovery spanning the genomic sequence of the locus and high-density linkage disequilibrium (LD) mapping. The meta-analysis provided evidence for association between rs1344706 that surpasses widely accepted benchmarks of significance by several orders of magnitude for both schizophrenia (P=2.5 × 10−11, odds ratio (OR) 1.10, 95% confidence interval 1.07–1.14) and schizophrenia and bipolar disorder combined (P=4.1 × 10−13, OR 1.11, 95% confidence interval 1.07–1.14). After de novo polymorphism discovery and detailed association analysis, rs1344706 remained the most strongly associated marker in the gene. The allelic association at the ZNF804A locus is now one of the most compelling in schizophrenia to date, and supports the accumulating data suggesting overlapping genetic risk between schizophrenia and bipolar disorder.

Meta-analysis of 32 genome-wide linkage studies of schizophrenia

A genome scan meta-a nalysis (GSMA) was carried out on 32 independent genome-wide linkage scan analyses that included 3255 pedigrees with 7413 genotyped cases affected with schizophrenia (SCZ) or related disorders. The primary GSMA divided the autosomes into 120 bins, rank-ordered the bins within each study according to the most positive linkage result in each bin, summed these ranks (weighted for study size) for each bin across studies and determined the empirical probability of a given summed rank (PSR) by simulation. Suggestive evidence for linkage was observed in two single bins, on chromosomes 5q (142–168 Mb) and 2q (103–134 Mb). Genome-wide evidence for linkage was detected on chromosome 2q (119–152 Mb) when bin boundaries were shifted to the middle of the previous bins. The primary analysis met empirical criteria for ‘aggregate’ genome-wide significance, indicating that some or all of 10 bins are likely to contain loci linked to SCZ, including regions of chromosomes 1, 2q, 3q, 4q, 5q, 8p and 10q. In a secondary analysis of 22 studies of European-ancestry samples, suggestive evidence for linkage was observed on chromosome 8p (16–33 Mb). Although the newer genome-wide association methodology has greater power to detect weak associations to single common DNA sequence variants, linkage analysis can detect diverse genetic effects that segregate in families, including multiple rare variants within one locus or several weakly associated loci in the same region. Therefore, the regions supported by this meta-analysis deserve close attention in future studies.

MicroRNA expression profiling in the prefrontal cortex of individuals affected with schizophrenia and bipolar disorders

MicroRNAs (miRNAs) are a large family of small non-coding RNAs which negatively control gene expression at both the mRNA and protein levels. The number of miRNAs identified is growing rapidly and approximately one-third is expressed in the brain where they have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. Here we investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N = 35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as being differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. In an initial attempt to corroborate some of these predictions, we investigated the extent of correlation between the expressions of hsa-mir-34a, -132 and -212 and their predicted gene targets. mRNA expression of tyrosine hydroxylase (TH), phosphogluconate dehydrogenase (PGD) and metabotropic glutamate receptor 3 (GRM3) was measured in the SMRI sample. Hsa-miR-132 and -212 were negatively correlated with TH (p = 0.0001 and 0.0017) and with PGD (p = 0.0054 and 0.017, respectively).

Replication of association between schizophrenia and ZNF804A in the Irish Case-Control Study of Schizophrenia sample

A recent genome-wide association study reported association between schizophrenia and the ZNF804A gene on chromosome 2q32.1. We attempted to replicate these findings in our Irish Case–Control Study of Schizophrenia (ICCSS) sample (N=1021 cases, 626 controls). Following consultation with the original investigators, we genotyped three of the most promising single-nucleotide polymorphisms (SNPs) from the Cardiff study. We replicate association with rs1344706 (trend test one-tailed P=0.0113 with the previously associated A allele) in ZNF804A. We detect no evidence of association with rs6490121 in NOS1 (one-tailed P=0.21), and only a trend with rs9922369 in RGRIP1L (one-tailed P=0.0515). On the basis of these results, we completed genotyping of 11 additional linkage disequilibrium-tagging SNPs in ZNF804A. Of 12 SNPs genotyped, 11 pass quality control criteria and 4 are nominally associated, with our most significant evidence of association at rs7597593 (P=0.0013) followed by rs1344706. We observe no evidence of differential association in ZNF804A on the basis of family history or sex of case. The associated SNP rs1344706 lies in ∼30 bp of conserved mammalian sequence, and the associated A allele is predicted to maintain binding sites for the brain-expressed transcription factors MYT1l and POU3F1/OCT-6. In controls, expression is significantly increased from the A allele of rs1344706 compared with the C allele. Expression is increased in schizophrenic cases compared with controls, but this difference does not achieve statistical significance. This study replicates the original reported association of ZNF804A with schizophrenia and suggests that there is a consistent link between the A allele of rs1344706, increased expression of ZNF804A and risk for schizophrenia.

Molecular genetic studies of schizophrenia.

The study of schizophrenia genetics has confirmed the importance of genes in etiology, but has not so far identified the relationship between observed genetic risks and specific DNA variants, protein alterations or biological processes. In spite of many limitations, numerous regions of the human genome give consistent, although by no means unanimous, support for linkage, which is unlikely to occur by chance. Two recent shifts have been evident in the field. First, a series of studies combining linkage and association analyses in the same family sets have identified promising candidate genes (DTNBP1, NRG1, G72/G30, TRAR4). Although a consensus definition of replication for genetic association in a complex trait remains difficult to achieve, the evidence for two of these (dystrobrevin binding protein 1 (DTNBP1), NRG1) is strong. Second, a series of studies combining association with functional investigation of changes in the associated gene in schizophrenia have also identified several candidate genes (COMT, RGS4, PPP3CC, ZDHHC8, AKT1). Somewhat surprisingly, the loci implicated by these studies have proven less robust in replication, although the number of replication studies remains small in several cases. Assessment of the combined evidence for the DTNBP1 gene gives some insight into the nature of the problems remaining to be solved.

Linkage and associated studies of schizophrenia

Genetic epidemiology has provided consistent evidence over many years that schizophrenia has a genetic component, and that this genetic component is complex, polygenic, and involves epistatic interaction between loci. Molecular genetics studies have, however, so far failed to identify any DNA variant that can be demonstrated to contribute to either liability to schizophrenia or to any identifiable part of the underlying pathology. Replication studies of positive findings have been difficult to interpret for a variety of reasons. First, few have reproduced the initial findings, which may be due either to random variation between two samples in the genetic inputs involved, or to a lack of power to replicate an effect at a given alpha level. Where positive data have been found in replication studies, the positioning of the locus has been unreliable, leading no closer to positional cloning of genes involved. However, an assessment of all the linkage studies performed over the past ten years does suggest a number of regions where positive results are found numerous times. These include regions on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10, 13, 15, 18, 22 and the X. All of these data are critically reviewed and their locations compared. Reasons for the difficulty in obtaining consistent results and possible strategies for overcoming them are discussed. Am. J. Med. Genet. (Semin. Med. Genet.) 97:23-44, 2000.

Identification of a high-risk haplotype for the dystrobrevin binding protein 1 (DTNBP1) gene in the Irish study of high-density schizophrenia families.

A recent report showed significant associations between several SNPs in a previously unknown EST cluster with schizophrenia.1 The cluster was identified as the human dystrobrevin binding protein 1 gene (DTNBP1) by sequence database comparisons and homology with mouse DTNBP1.2 However, the linkage disequilibrium (LD) among the SNPs in DTNBP1 as well as the pattern of significant SNP–schizophrenia association was complex. This raised several questions such as the number of susceptibility alleles that may be involved and the size of the region where the actual disease mutation(s) could be located. To address these questions, we performed different single-marker tests on the 12 previously studied and 2 new SNPs in DTNBP1 that were re-scored using an improved procedure, and performed a variety of haplotype analyses. The sample consisted of 268 Irish multiplex families selected for high density of schizophrenia. Results suggested a simple structure where the LD in the target region could be explained by 6 haplotypes that together accounted for 96% of haplotype diversity in the whole sample. From these six, a single high-risk haplotype was identified that showed a significant association with schizophrenia and explained the pattern of significant findings in the analyses with individual markers. This haplotype was 30 kb long, had a large effect, could be measured with two tag SNPs only, had a frequency of 6% in our sample, seemed to be of relatively recent origin in evolutionary terms, and was equally distributed over Ireland. Implications of these findings for follow-up and replication studies are discussed.